The long term goal of this project is to understand the roles of cell surface heparan sulfate in contributing herpes simplex viral infection. Heparan sulfate is a highly sulfated polysaccharide with very complicated saccharide sequences, and is present on the mammalian cell surface and in the extracellular matrix in a large quantity. Although heparan sulfate is a known important cell-surface molecule involved in assisting herpes virus infection for a long time, the relationship between the saccharide structure and its role in assisting herpes viral infection is poorly understood. We propose to conduct a series of biochemical studies to elucidate the structural specificity of the 3-O-sulfated heparan sulfate, which is generated by three different heparan sulfate 3-O-sulfotransferase (3-OST) isoforms, for the binding to herpes envelope glycoprotein D (gD). In particular, we plan to carry out the following projects: 1. Isolation and characterization of the gD-binding oligosaccharides generated by isoform 3 (3-OST-3). We plan to prepare the gD-binding oligosaccharide by incubating purified 3-OST-3 enzyme with a heparan sulfate oligosaccharide library. The gD-binding oligosaccharide will be purified using anion exchange HPLC and gD-affinity column. The structure of the gD-binding oligosaccharide will be determined by chemical and enzymatic degradation approaches coupled with matrix assisted laser desorption/ionization mass spectrometry. We also plan to examine the effect of the purified gD-binding oligosaccharide on viral entry into the cell using a cell-based assay. 2. Characterization of the structures of the gD-binding sites generated by isoform 2 and isoform 4 (3-OST-2 and 3-OST-4). We plan to express and purify 3-OST-2 and 3-OST-4 enzymes. We will also determine the structures of the gD-binding sites within 3-OST-2 and 3-OST-4 modified heparan sulfate. Both 3-OST-2 and 3-OST-4 have recently proved to assist herpes simplex virus 1 entry into the cells, suggesting that 3-OST-2 and 3-OST-4 provide binding sites for gD. In addition, studies of the distribution of 3-OST-2 and 3-OST-4 revealed that both enzymes are highly expressed in human brains. We speculate that herpes virus may utilize 3-OST-2 and 3-OST-4 modified heparan sulfate to infect human brains.